Feeding behavior is critical for animal survival, and is also a fundamental aspect of energy homeostasis. This process is regulated by a highly complex neuroendocrine system which involves a multitude of neuropeptides and amines. Despite decades of work on individual neurotransmitter or peptidergic signaling systems, the general organizational principles underlying neuromodulation are still poorly understood. This is, in part, due to a lack of analytical capabilities to measure and identify these low abundance endogenous signaling molecules in a complex microenvironment. The long term goal of our research is to develop new bioanalytical methods to elucidate the complex identities and functional roles of neuropeptides in food intake and to expand our fundamental understanding of cotransmission and neuromodulation at the molecular level. During our previous grant funding period, considerable progress has been made leading to the discovery of more than 200 novel neuropeptides with several neuropeptide families consisting of as many as 20-40 members in a simple crustacean model system. This stunning chemical complexity coupled with its best characterized neuronal circuit offers an unprecedented opportunity to investigate the intriguing question whether these individual variants play distinct roles in regulation of food intake. To address significant biological questions related to functional significance of peptide multiplicity and diversity, we propose the development and application of new analytical methodologies and capabilities using both the crustacean and mammalian nervous systems. The specific aims of the proposal include: (1) To develop a MALDI-based mass spectral imaging (MSI) technique for mapping co-localization patterns of individual isoforms of extended peptide families and amine neurotransmitters in identified neurons and the feeding circuits. A set of novel multiplexed isobaric labeling reagents will be incorporated for quantitative assessment of neuropeptide expression upon feeding; (2) To develop a nanoparticle-based affinity-enhanced microdialysis in vivo sampling technique coupled with monolithic microscale separation for MS detection and quantitation of secreted neuropeptides in response to food intake; (3) To determine the functional consequences of neuropeptide isoforms via a combination of mass spectrometric and electrophysiological techniques. Individual variants of major peptide families involved in food intake will be investigated for their differential degradation profiles and distinct physiological actions on the feeding circuits. The outcome of the proposed research will be the development of innovative methodologies for probing neurochemistry at the cellular and network levels and an improved understanding of peptide multiplicity in regulation of food intake and other physiological functions. The parallel application of these new methods to both crustacean and mammalian nervous systems in feeding will accelerate our pace towards the development of new therapeutics for feeding disorders.